Quality Control Requirements

The CLIA quality control requirements are found in Subpart K of the Code of Federal Regulations. Every laboratory must establish and follow written quality control procedures designed to monitor and evaluate the quality of the testing process for each method to assure accurate and reliable patient test results.

General Requirements

For moderately and highly complex tests, the laboratory must:

Follow the manufacturer's instructions.
Have a procedure manual describing the process of the tests and reporting patient test results.
Perform and document calibration procedures or check calibration at least once every six months.
Perform and document control procedures using at least two levels of controls each day of testing.
Perform and document applicable specialty and subspecialty control procedures.
Perform and document remedial action taken when problems or errors are identified.
Maintain records of all quality control activities for two years (five years for immunohematology).

Facilities

The laboratory must have space and environmental conditions necessary for conducting the services offered. This includes being constructed, arranged and maintained to ensure the space, ventilation and utilities necessary for conducting all phases of testing. Safety precautions must be established, posted and observed to ensure protection from physical, chemical, biochemical and electrical hazards and biohazardous materials.

Test Methods, Equipment, Instruments, Reagents, Materials and Supplies

The laboratory must utilize test methods, equipment, instruments, reagents, materials and supplies that provide accurate and reliable test results and reports. Requirements include:

Selecting methodologies and equipment and performing testing in a manner that provides test results within the laboratory's stated performance specifications for each method.
Utilize appropriate and sufficient equipment, instruments, reagents, materials and supplies for the type and volume of testing performed and for the assurance of quality throughout the testing process.
Define criteria for: storing reagents and specimens; accurate and reliable test system operation; and, test result reporting, including (as applicable):
- Water quality
- Temperature
- Humidity
- Protection of equipment and instrumentation from fluctuations and interruptions in electrical current that adversely affect results and reports.
- Document remedial actions implemented to correct conditions that fail to meet criteria.
Label reagents, solutions, culture media, control materials, calibration materials and other supplies including identification for:
- Storage requirements
- Identity, and when pertinent, titer, strength or concentration
- Preparation and expiration date
- Other appropriate information
Prepare, store and handle reagents, solutions, culture media, control materials, calibration materials and other supplies in a manner to ensure:
- Items are not used when they have exceeded their expiration date, have deteriorated or are of sub-standard quality.
- Components of reagent kits of different lot numbers are not interchanged unless otherwise specified by the manufacturer.

Procedure Manual

The laboratory must have a written procedure manual for the performance of all analytical methods used by the laboratory. The manual must be readily available and followed by laboratory personnel. Textbooks may be used as a supplement but not in place of the procedure manual. The following information is required to be included:

Specimen collection, processing and rejection criteria requirements.
Procedures for microscopic examinations, including the detection of inadequately prepared slides.
Step-by-step performance of the procedure, including test calculations and interpretation of results.
Preparation of slides, solutions, calibrators, controls, reagents, stains and other materials used in testing.
Calibration and calibration verification procedures
The reportable range of patient test results as established or verified through method performance specifications.
Control procedures
Remedial action to be taken when calibration or control results fail to meet the laboratory's criteria for acceptability.
Limitations in methodologies, including interfering substances.
Reference or normal ranges
Imminent life-threatening laboratory results or panic (critical) values.
Pertinent literature references
Appropriate criteria for specimen storage and preservation to ensure specimen integrity until testing is completed.
The laboratory's system for reporting patient results including, when appropriate, the protocol for reporting panic values.
Description of the steps to be taken in the event that a test system becomes inoperable.
Criteria for the referral of specimens including procedures for specimen submission and handling.

Establishment and Verification of Method Performance Specifications

Prior to beginning to report patient results, the laboratory must establish and verify (for all tests and methods) performance specifications, including:

Accuracy
Precision/Analytical Sensitivity
Analytical Specificity

If applicable, the laboratory must also verify the reportable range of patient results as well as the reference (normal) range. When a new procedure is implemented, the laboratory must demonstrate, prior to reporting patient results, that it can obtain the performance specifications comparable to the manufacturer's established specifications. The laboratory must also verify the manufacturer's reference range for the laboratory's population. The laboratory must then establish calibration and quality control procedures based on the verified performance specifications. Each step must be documented in the laboratory's records.

Equipment Maintenance and Function Checks

The laboratory is required to perform equipment maintenance and function checks (electronic, mechanical and operational). These checks are considered to be necessary for proper test performance and result reporting to assure accurate and reliable test results and reports. The requirements for systems approved by the FDA include maintenance performance as specified by the manufacturer and with at least the frequency specified by the manufacturer. All maintenance performed by the laboratory should be documented. The same applies to system function checks - follow the manufacturer's instructions for both the checks and frequency and document each step.

Calibration and Calibration Verification

The laboratory is required to substantiate the continued accuracy of each of its test methods, throughout its reportable range for patients, through calibration and calibration verifications.

The reportable range of patient test results is the range of test result values over which the laboratory can establish or verify the accuracy of the instrument, kit or test system measurement response.
Calibration is the process of testing and adjusting an instrument, kit or test system to provide a known relationship between the measurement response and the value of the substance that is being measured by the test procedure.
Calibration verification is the assaying of calibration materials in the same manner as patient samples to confirm that the calibration of the instrument, kit or test system has remained stable throughout the laboratory's reportable range for patient test results.

The manufacturer's instructions should be followed, along with using the materials specified by the manufacturer. Each step should be documented.

Control Procedures

On a routine basis, the laboratory must perform control procedures to monitor the stability of the methods or systems utilized by the laboratory. Control and calibration materials indirectly assess the accuracy and precision of patient test results. At a minimum, the manufacturer's instructions are to be followed. The laboratory must:

Test quality control samples in the same fashion as patient specimens.
Determine the statistical parameters (e.g., mean , standard deviation) for each lot number through repetitive testing. The stated values of an assayed control material may be used as the target values provided the stated values correspond to the methodology and instrumentation employed by the laboratory and are verified by the laboratory.
Accept control results only when the laboratory's criteria for acceptability is met.
Conduct reagent and supply checks for each shipment or batch of reagents, discs, stains, antiserum and identification system when opened. The checks should include positive and negative reactivity, as well as graded reactivity, if applicable.
The laboratory must test staining material each day of use to ensure predicted staining characteristics.
For microbiology media, the laboratory must check for sterility, ability to support growth and, as applicable, selectivity/inhibition and/or biochemical response. The laboratory may use the manufacturer's control checks provided the manufacturer's checks meet the National Committee for Clinical Laboratory Standards (NCCLS) for media quality control.
Document the physical characteristics of the media to confirm that the media has not been compromised. Report deterioration to the manufacturer.
Follow the manufacturer's specifications for using the media.

Remedial Actions

The laboratory must establish policies and procedures for remedial actions for quality control failures and apply them as needed to maintain accurate and reliable patient test results and reports. The laboratory must document when:

Test systems do not meet the established performance specifications. Examples include when equipment or methodologies perform outside established parameters and when patient results are outside of the reportable range.
Control or calibration results fail to meet established criteria. When this occurs, patient results tested between the previous acceptable and the current unacceptable run must be evaluated to determine if the patient results had been affected. The laboratory must take remedial action to ensure the reporting of accurate and reliable results.
The laboratory cannot report patient test results within its regular time frames. The laboratory must determine (based on the urgency of the tests requested) the need to notify the appropriate individual of the delay.
Errors are detected in reported patient test results.
- The laboratory must promptly notify the authorized person ordering the test or the individual utilizing the test results.
- The laboratory must issue corrected reports promptly to the authorized person ordering the test or the individual utilizing the test results.
- The laboratory must maintain exact duplicates of the original and corrected report for two years.

QC Record Retention Requirements
Laboratory Record	Period of Retention
Discontinued procedures	2 years
Method Performance Specifications	2 years
Equipment Maintenance and Function Checks	2 years
Calibration and Calibration Verification	2 years
Control Procedures (e.g., daily QC records)	2 years
Remedial Action - errors in reported results (both the original and corrected report)	2 years

Specialty/Subspecialty Requirements

Along with meeting the general requirements, laboratories must also meet the following specialty/subspecialty requirements.

Bacteriology

The laboratory must check positive and negative reactivity with control organisms:

Each day of use for catalase, coagulase, beta-lactamase and oxidase reagents and DNA probes.
Each week of use for Gram and acid-fast stains, bacitracin, optochin, ONPG, X and V discs or strips.
Each month of use for antisera.
Each week of use for XV discs or strips (with a positive control organism).
Each new batch of media and each lot of antimicrobial discs before or concurrent with the initial use, using approved reference organisms. Zone sizes or minimum inhibitory concentration for reference organisms must be within established limits prior to reporting patient results. Each day of use, test appropriate control organisms to check the procedure.

General Immunology

The laboratory is required to:

Run serologic tests on patient specimens concurrently with positive control serum of known titer or controls of graded activity (if applicable), along with a negative control.
Utilize controls that evaluate all phases of the test system to ensure reactivity and uniform dosages when positive and negative controls alone are not sufficient.
Not report result unless the predetermined reactivity pattern is observed with the controls.

Hematology

The laboratory is required to:

Perform in duplicate manual cell counts (using a hemocytometer). One control is required for each eight hours of testing
Include two levels of controls for each eight hours of operation for non-manual hematology systems.
Include two levels of controls for each eight hours of operation for non-manual coagulation systems. Two levels of controls must be tested each time reagent is changed.
Include two levels of control for each individual performing manual coagulation tests before testing patient specimens and each time reagent is changed.
Test patient and control specimens in duplicate when performing manual coagulation testing.

Quality Control - General Information

Quality control procedures are a tool to detect problems that could invalidate patient results. Quality control alerts the testing personnel that the test performance could be better if a problem is developing with the test procedure. Quality control specimens have known values that are run along with the patient samples. If the result for the control sample is within the acceptable range, it is assumed that the values for the patient samples are also reliable.

The laboratory quality control program should be a monitoring system that, first, will provide immediate information for making the decision about the acceptability of patient results. Second, the quality control program should easily provide a method for evaluating data over time to help in making decisions about the overall performance of the test procedure.

As mentioned, a control is a specimen with a known value or range of values that is tested like a patient sample in order to check the accuracy and precision of a testing method. These controls should be run on both qualitative (result is positive or negative) and quantitative (result is a number or value) tests. While the control sample is treated as a patient sample during the testing process, it is not collected, transported, or always stored like a patient sample. Some quantitative controls may require reconstitution that would add another variable unlike the patient sample.

Qualitative Test

A qualitative test may have a built in positive control based on a certain level of detection. An example of a qualitative test is the pregnancy test. This type of test has a built-in positive control. If the positive control is not positive as it should be then the test should be repeated. If further investigation is needed, there are several possible causes for an incorrect control in a qualitative test. First, the reagents may be added in wrong order. Second, the test procedure may be done correctly, but the results might be read too soon or too late. Also, the reagents could be outdated or possibly have deteriorated from inadequate storage. Document that there is a problem and list the QC code and expiration date. If everything seems to be accurate and the problems are still evident, then talk to the kit manufacturer about the problem.

Quantitative Test

Quantitative tests have been simplified in the last few years due to the increased stability of reagents and instruments. However, quality control records should be kept to allow laboratory or office lab staff to monitor the test's performance and recognize problems.

Quality Control Terms

The mean is determined by adding a group of measured values, then dividing the total by the number of measurements in the group. The standard deviation is determined by first calculating the mean, then taking the difference of each control result from the mean, squaring that difference, dividing by n-1, then taking the square root. It is easy to use a scientific calculator, an electronic spreadsheet, or a statistics program, all of which have functions for calculating the standard deviation of a group of measurements. The coefficient of variation (C.V.) describes the standard deviation as a percent of the mean. C.V. is calculated by dividing the S.D. by the mean and multiplying by 100 percent. While the S.D. changes with concentration, i.e., the larger the concentration, the larger the standard deviation; the C.V. reflects a ratio of the S.D. to the concentration. In the clinical laboratory, the C.V. is useful in comparing the precision of a variety of determinations that have different normal values and even different units as well. After a statistically significant number of control determinations have been completed, usually 20 or more, the standard deviation and the coefficient variation are then calculated. It is common practice to calculate the coefficient of variation (C.V.) on a monthly basis.

Statistically, if there is a normal distribution of the control values we would have approximately 68% of the points within 1 Standard Deviation (S.D.) of the Mean, 95% within 2 Standard Deviations (S.D.) of the Mean and 99.7% within 3 Standard Deviations (S.D.) of the Mean. Standard deviation is the distribution of control results around the expected mean.

Investigating Quality Control Errors

Statistically, one in twenty of your values may fall outside of the acceptable limits; however, when a result exceeds the limits, reanalyze the same control immediately. Record both control values if the reanalyzed control now falls within acceptable limits, and the patient results may now be reported. If one or both of the control values still fall outside acceptable limits, further investigation may be warranted. First, reconstitute a new vial of control or assay a new prediluted control vial. The next step is to check routine instrument maintenance procedures and then recalibrate the instrument according to the manufacturer. If the problem persists, call the instrument manufacturer for further troubleshooting information.

What should you look for on your QC charts? Check for systematic errors that are continuous and affect all results. Systematic errors can be due to failure of materials, procedural mistakes, or personnel errors. If the system is out of control, no further patient testing should be done until the error has been determined and corrected.

The problems that will be evident on the QC charts are excessive scatter, test bias, drifting of results, shifting of results or a trend. First, “excessive scatter” is actually poor reproducibility of results. The points on the chart are widely scattered above and below the mean. The most common cause for this is operator variability, which may be due to variability in pipetting or possibly inattention to critical steps in the procedure. See example of a glucose control chart below.

Next, “bias” with the test results means that the majority of the points fall either above or below the mean. The two most common causes for bias are an error in calibration or an error in reconstituting a control solution. See example of a glucose control chart below.

The third problem with QC results may be demonstrating “drift” or a “trend” which occurs when the QC results move away from the mean in a particular direction over an extended period of time. This may occur when reagents are outdated or have gradually deteriorated or if the instrument is losing its calibration. See example below.

The QC chart may demonstrate a “shift” when the controls suddenly change from moving nicely back and forth above and below the mean to a line that is either always above the mean or always below the mean. The most common causes of a “shift” in QC are: recalibration of the instrument was just performed; a new lot number of reagents has been put into use; a sudden deterioration of the reagents (possibly left out of the refrigerator overnight); a new person is performing the testing; or even possibly a major change in the instrument performance. See example below.